Identification of cis-elements and trans-acting factors involved in fatty acid-mediated regulation of OLE1 mRNA stability in Saccharomyces cerevisiae.

摘要

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta 9 fatty acid desaturase, an enzyme that converts saturated acyl-coenzyme A precursors into monounsaturated fatty acids. OLE1 gene expression is repressed in response to nutritional and physiological conditions through transcriptional and post-transcriptional controls that include a mechanism that regulates OLE1 mRNA stability. OLE1 mRNA is a moderately stable species with a half-life of 10 +/- 1.5 minutes (basal stability) under normal growth conditions in fatty acid free medium. Its half-life drops to 2 +/- 1.5 minutes when cells are exposed to unsaturated fatty acid for 15 minutes (regulated stability). This thesis describes the identification of cis elements and a trans-acting factor that are involved in the fatty acid mediated regulation of OLE1 mRNA stability. OLE1 produces three primary transcripts that differ with respect to the length of their 5' untranslated region (UTR). The longest transcript has a >220 nucleotide 5' UTR, while the shorter transcripts contain 70 and 55 base 5 'UTR elements. Deletion and substitution of sequences in the OLE1 transcript show that nucleotides from -207 to -70 in the 5'UTR, sequence elements that encode for both the transmembrane loops of Ole1p and sequences within the cytochrome b5 functional domain appear to be essential for regulated mRNA stability. Experiments performed to determine whether the nucleotide or polypeptide sequence encoded by the critical cis elements are important for stability regulation by introducing stop codons at various positions to abort the synthesis of Ole1p show that the mRNA sequence elements and not the Ole1p is essential for regulation. Replacing OLE1 3'UTR with the 3'UTR derived from the highly stable PGK1 gene did not affect the basal stability of the transcript although it did slightly reduce the range of unsaturated fatty acid mediated regulation. OLE1 mRNA stability regulation was found to be dependent on Mga2p, a proteosome cleavable ER membrane protein. In mga2Delta deletion strain, the half-life under fatty acid free and fatty acid supplemented conditions are 4 and 5 minutes respectively, showing the loss of regulation. Surprisingly SPT23, which is a homolog of MGA2, does not appear to be involved in fatty acid mediated regulation of OLE1.